Innate immunity plays a crucial role in initiating the immune responses and pathogen clearance upon microbial infection. Using pattern recognition receptors including cell surface receptor such as Toll-like receptors (TLRs) or intracellular receptors such as NOD-like receptors (NLRs), host cells sense and respond to microbial infection, which triggers the activation of various signaling events, leading to anti-microbial and inflammatory responses. Understanding the signaling pathways induced by various microbial infections will provide the molecular insights into the host defense machinery and provide the potential therapeutic targets for anti-microbial infection. Recent studies from our lab and others indicate that CARD9, a Caspase Recruitment Domain (CARD)-containing adaptor protein, plays an important role in innate immune responses through regulation of NF-kB and MAP kinases. Interestingly, we have found that CARD9 physically associates with bacteria-containing phagosomes and is directly involved in the clearance of infected bacteria in macrophages. However, the molecular mechanism by which CARD9 is involved in the regulation of these responses remains to be determined. To further study the biological function of CARD9, we propose two specific aims to investigate the molecular mechanism by which CARD9 mediates antimicrobial responses using our recently generated CARD9-deficient (Card9-/-) mice. Our specific aims are: (1) to determine the molecular mechanism by which CARD9 is involved in the clearance of intracellular bacteria; (2) to identify CARD9-interacting proteins that mediate CARD9-dependent anti-bacterial and fungal responses. Together, these studies will reveal an unknown host defense mechanism and provide the novel insight into anti-microbial immune responses.